Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 4. ARID1A deletion renders HCC cells resistant to glucose deprivation via activation of the AMPK pathway. The effect of ARID1A knockout on Huh7 and YY-8103 cells upon glucose starvation is investigated by (A) Annexin V–fluorescein isothiocyanate (FITC)/PI apoptosis kit, and (B) the result of quantitative analysis is shown. (C) The expression of AMPK signaling proteins in liver tissues from control and Arid1a liver-specific knockout mice. (D) The expression of the indicated proteins in AMPK signaling in primary hepatocytes from control and Arid1a liver-specific KO mice. The expression of (E) PRKAA2 in control, ARID1A knockout YY-8103, Huh7 cells and (F) ARID1A-overexpressing PVTT and SNU-398 cells. The mRNA level of (G) Prkaa1 and (H) Prkaa2 in liver tissues from control and Arid1a liver-specific knockout mice. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05, ***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activation Assay, Knock-Out, Expressing, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 6. ARID1A regulates the ubiquitination of PRKAA2 through USP9X. The influence of ARID1A on the ubiquitination of PRKAA2 in (A) HEK293T, (B and C) SNU-398, PVTT, and (D) Huh7 cells. (E) The mRNA level of proteins involved in PRKAA2 ubiquitination or deubiquitination in liver tissues from control and Arid1a liver-specific knockout mice. (F) The mRNA level of USP9X in control and ARID1A KO Huh7 (left) and YY-8103 (right) cells is examined by real-time PCR. (G) Usp9x expression in liver tissues from control and Arid1a liver-specific knockout mice is examined by Western blot. (H) USP9X expression in control and ARID1A KO Huh7 and YY-8103 cell is examined by Western blot. (I) USP9X expression in control and ARID1A- overexpressing SNU-398 cells is examined by Western blot. (J) USP9X and PRKAA2 expressions in control and ARID1A KO MHCC97H cells are examined by Western blot. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05,**P<0.01,***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Ubiquitin Proteomics, Control, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 9. ARID1A regulates the promoter activity of USP9X via HDAC1. (A) Data from the Catalogue of Somatic Mutations in Cancer shows that 1989* is the most frequent mutation of ARID1A. (B) Interaction between ARID1A-WT or ARID1A-1989* mutation with HDAC1. (C) Influence of ARID1A-WT or ARID1A-1989* mutation on the ubiquitination of PRKAA2. (D) Influence of ARID1A-WT or ARID1A-1989* mutation on the promoter activity of USP9X. The promoter activity of USP9X in (E) HEK293T cells overexpressing ARID1A or HDAC1 (OE) or in (F) ARID1A knockout Huh7 and YY-8103 cells is examined by luciferase reporter assay. (G) Influence of ARID1A-WT or ARID1A-1989* mutation on the expression of USP9X and PRKAA2. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **P<0.01, ***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activity Assay, Mutagenesis, Ubiquitin Proteomics, Knock-Out, Luciferase, Reporter Assay, Expressing, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 11. ARID1A negatively correlates with USP9X/PRKAA2 and influences HCC patients’ survival. The correlation among ARID1A, USP9X, and PRKAA2 in the clinical samples is examined by (A) Western blot or by (B) immunohistochemical staining in the Human Protein Atlas (HPA) database. (C) Immunohistochemistry staining of ARID1A, USP9X, and PRKAA2 in HCC tissues in TMAs. Scale bar: 100 mm. (D) The correlation between USP9X and PRKAA2 at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (E) The correlation between ARID1A and USP9X at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (F) Comparison of overall survival between HCC patients with different ARID1A/ USP9X expressions. Data are analyzed using the log-rank test. (G) The correlation between ARID1A and PRKAA2 at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (H) Comparison of overall survival between HCC pa- tients with different ARID1A/PRKAA2 expressions. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry, Comparison

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 14. The effects of inactivation of PRKAA2 and USP9X on HCC cell growth. (A) The growth of control (Scramble, SCR) and PRKAA2 knockdown (sh1#, sh2#) cells is measured by crystal violet staining under both normal and glucose-deprived conditions. The influences of (B, D, E) Compound C and (C, F, G) WP1130 on HCC cell growth is measured by crystal violet staining or cell counting kit-8 (CCK8) assay under both normal and glucose- deprived conditions.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Control, Knockdown, Staining, Cell Counting, CCK-8 Assay

Journal: Cell Reports

Article Title: Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis

doi: 10.1016/j.celrep.2022.110761

Figure Lengend Snippet:

Article Snippet: pCMV6-myc-DDK-AMPK-α1 with C-terminal DDK tag (RC218572), pCMV6-myc-DDK-AMPK-α2 with C-terminal DDK tag (RC210226), and pCMV6-AN-Myc-DDK-AMPK-α1 with N-terminal DDK tag (RC210226) were purchased from OriGene Technologies, Inc (Rockville, MD, USA). pRP[Exp]-Puro-EF1A>mCherry-AMPK-α1 with N-terminal mCherry tag (VB180924-1121rhh), pRP[Exp]-Puro-EF1A>EGFP-AMPK-α1 with N-terminal GFP tag (VB180924-1122ycm), pRP[Exp]-Puro-EF1A>EGFP control vector (VB180823-1120qay), pRP[Exp]-Puro-EF1A>EGFP-AMPK-α1 (aa1-529) with N-terminal GFP tag (VB180924-1125vax), and pRP[Exp]-Puro-EF1A>AMPK-α1(aa530-559)-EGFP (VB180924-1128rtd) with C-terminal GFP tag were purchased from Vector Builder Inc. (Chicago, IL, USA).

Techniques: Recombinant, Protease Inhibitor, Transfection, Expressing, Integrity Assay, Mutagenesis, Single Cell Gel Electrophoresis, Plasmid Preparation, Software

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet: PHBs are negative regulators of AMPK, related to Figures S3 and (A) HepG2 cells transfected with control (siCtrl) or PHB1 and PHB2 (siPHB1/2) siRNAs were subjected to immunoblot analysis with antibodies to the indicated proteins. The p-AMPKα/AMPKα, p-ACC/ACC, PHB1/β-actin, and PHB2/β-actin ratios were determined by densitometry and are expressed relative to the corresponding mean value for siCtrl-transfected cells. (B) HepG2 cells transfected with siCtrl or siPHB1/2 were incubated with [ 14 C]acetate and then assayed for fatty acid synthesis. (C) Immunoblot analysis of a whole cell extract (WCE) as well as of isolated mitochondrial (Mito) and cytosolic fractions of HepG2 cells with antibodies to AMPKα, to PHB1, to PHB2, to HSP90 (cytosolic marker), and to COX IV (mitochondrial marker). (D) Mitochondrial lysates prepared from HepG2 cells were subjected to immunoprecipitation (IP) with antibodies to AMPKα or with control immunoglobulin G (IgG), and the resulting precipitates as well as the original lysates (Input) were subjected to immunoblot analysis with antibodies to the indicated proteins. (E) Recombinant GST-PHB1 or GST alone, each bound to glutathione-agarose beads, was incubated with recombinant His 6 -tagged AMPK (α2β1γ1) in the absence or presence of RX-375 (2 μM), after which bead-bound proteins were subjected to immunoblot analysis with antibodies to the indicated proteins. (F) HepG2 cells expressing 3×Flag-tagged PHB1 and Myc epitope-tagged AMPKα2 were incubated with RX-375 (1 μM) for 3 h, after which cell lysates were subjected to immunoprecipitation with antibodies to Flag and the resulting precipitates were subjected to immunoblot analysis with antibodies to Myc and to Flag. All quantitative data are means ± s.e.m. from five (B) or six (A) independent experiments (n = 5 or 6, respectively). ∗p < 0.05 versus siCtrl, unpaired two-tailed Student’s t test (A and B).

Article Snippet: In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm).

Techniques: Transfection, Control, Western Blot, Incubation, Isolation, Marker, Immunoprecipitation, Recombinant, Expressing, Two Tailed Test

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm).

Techniques: Produced, FLAG-tag, Control, Recombinant, Membrane, Transfection, Reverse Transcription, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software

Journal: Autophagy

Article Title: AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

doi: 10.4161/auto.20586

Figure Lengend Snippet: Figure 6. AMPK regulates YWHAZ binding to ULK1. COS7 cells infected with myc-ULK1 were treated with (A) 25 mM 2DG; (B) 1 mM AICAR; or (C) EBSS for the times indicated. The 0 min time point indicates treatment with vehicle for the longest timepoint. Cell lysates were incubated with GST-YWHAZ bound to glutathione sepharose beads or plain glutathione sepharose beads as a control. Immunoblots were probed with the antibodies indicated. (D) Wild-type MEFs and MEFs lacking the genes for both (PRKAA1−/−,PRKAA2−/−) or one of the PRKAA isoforms (PRKAA1−/− and PRKAA2−/−) were treated with vehicle (0 min time point) or 25 mM 2DG for 15 min and endogenous ULK1 was pulled down with GST-YWHAZ. The GST-tag alone served as a negative control. Western blot analysis was performed with the antibodies indicated. (E) MEFs stably overexpressing myc-ULK were treated with 25 mM 2DG as in (D) and endogenous YWHAZ was immunoprecipitated using a pan-14–3-3 antibody or nonspecific mouse IgG1 as a negative control. Western blot analysis was performed with the antibodies indicated. (F) Wild-type or nonphosphorylatable serine to alanine mutants of myc-ULK1 at four potential YWHAZ-binding sites, S467A, S494A, S555A and T659A as well as the S555A-T659A double mutant were stably overexpressed in COS7 cells were treated with 25 mM 2DG as in (D). Following lysis, myc-ULK1 variants were pulled down with GST or GST-YWHAZ. Western blot analysis was performed with the antibodies indicated.

Article Snippet: Mouse embryonic fibroblasts (MEFs) deficient in the genes encoding one or both isoforms of PRKAA and corresponding wild-type MEFs were provided by Dr. B. Viollet (INSERM U567; Paris, France) and described previously.

Techniques: Binding Assay, Infection, Incubation, Control, Western Blot, Negative Control, Stable Transfection, Immunoprecipitation, Mutagenesis, Lysis

Journal: Autophagy

Article Title: AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

doi: 10.4161/auto.20586

Figure Lengend Snippet: Figure 8. ULK1 and AMPK regulate the intracellular localization of ATG9. (A) Wild-type MEFs carrying an empty vector or ULK1−/− MEFs expressing either an empty vector (V) or myc-tagged wild-type (wt) ULK1 or the ULK1 S555A-S637A-T659A (“3A”) mutant were cultured in regular growth media (“full media”) or EBSS (“starvation”) for 3 h, and intracellular localization of endogenous ATG9 (red staining) was examined by immunoflourescence. Nuclei were counterstained with DAPI (blue). (B) Quantitation of ATG9-staining in the experiment shown in (A). Analysis was performed using Cellprofiler Image analysis software. Each data point represents one cell, horizontal bars and error bars indicate means with 95% confidence intervals. A minimum of 20 cells per cell line and treatment condition was analyzed. Statistical significance was determined by unpaired t-tests with Welch’s correction. *** indicates a nominal p-value < 0.001, n.s. indicates a nonsignificant nominal p-value > 0.05. The result shown are representative for 3 independent experiments. (C) Wild-type MEFs and MEFs lacking the genes for both catalytic PRKAA-subunits (PRKAA1−/−;PRKAA2−/−) were treated and examined for intracellular ATG9-localization as in (A). (D) Quantitation of ATG9-staining in the experiment shown in (C). Image analysis and statistics were performed as in (B). Nominal p-values of p < 0.001, p < 0.05 and p > 0.05 are denoted as ***, *, and n.s. (nonsignificant), respectively.

Article Snippet: Mouse embryonic fibroblasts (MEFs) deficient in the genes encoding one or both isoforms of PRKAA and corresponding wild-type MEFs were provided by Dr. B. Viollet (INSERM U567; Paris, France) and described previously.

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Cell Culture, Staining, Quantitation Assay, Software

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: MTA1 is upregulated in the tamoxifen resistant breast cancer cells. ( A ) Whole cell lysates and total RNA obtained from the tamoxifen resistant and their parental cells were subjected to western blotting (top) and qRT-PCR analysis (bottom). Expression was normalized using the ACTB gene. Data are presented as the mean fold change over control±SEM (n = 3). **, P< 0.01 and ***, P< 0.001. ( B ) The MCF7/TAMR-1 and the parental cells were transfected with si MTA1 and then treated with 1 μM 4OHT for the indicated time periods. The number of viable cells were counted using a hemocytometer. Cell numbers were presented as the mean±SEM from duplicate plates and data obtained from 1 of 3 independent experiments with similar results are presented. ***, P< 0.001. ( C ) The MCF7/TAMR-1 and the parental cells were transfected with si MTA1 for 48 h. At the end of incubation, 20 nM bafilomycin A 1 (Baf) was added at 1 h prior to examination of autophagy flux. ( D ) The MCF7/TAMR-1 and the parental cells were transfected with siRNAs for 48 h and then treated with 4OHT for an additional 24 h. The expression of LC3 and NBR1 was determined by western blotting. The LC3-II and NBR1 level was quantified from band intensity using imageJ and expressed as relative to the level in the parent cells without treatment. The values were normalized by the intensities of the corresponding TUBA band. Data are presented as the mean±SEM (n = 3). *, P< 0.05, **, P< 0.01 and ***, P< 0.001 (right). Quantification of NBR1 is shown in Fig. S2A.

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Control, Transfection, Incubation

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: MTA1 induces tamoxifen resistance in vitro and in vivo. ( A ) The MTA1-overexpressing MCF7 cells were established using the pLJM1 lentiviral vector system. Expression level of MTA1 protein was analyzed by western blotting. ( B ) The MTA1-overexpressing MCF7 cells and the control cells were treated with 1 μM 4OHT for the indicated time points. The number of viable cells were counted using a hemocytometer. Cell numbers were presented as the mean±SEM from duplicate plates and data obtained from 1 of 3 independent experiments with similar results are presented. ***, P< 0.001. ( C ) Female athymic nude mice were inoculated with the MTA1-overexpressing MCF7 cells. When tumor volume reached approximately 100 mm 3 , a tamoxifen or mock pellet was implanted subcutaneously. Five wk later, the xenograft tumors were harvested and tumor volume was measured (top). Representative tumor images are shown (bottom). Data were expressed as mean±SEM (n = 4 to 8) in each group. **, P< 0.01.

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: In Vitro, In Vivo, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: Autophagy is elevated in the MTA1-overexpressing MCF7 cells. ( A ) The MCF7 cells that stably expressed MTA1 were pretreated with 20 nM bafilomycin A 1 (Baf) for 0.5 h and then treated with 5 μM 4OHT for an additional one h prior to examination of autophagy flux. The expression of LC3 was determined by western blotting (left). The LC3-II level was quantified from band intensity using ImageJ and expressed as relative to the level in the control cells without treatment. Values were normalized by the intensities of the corresponding TUBA band. Data are presented as the mean±SEM (n = 3). *, P< 0.05 and ***, P< 0.001 (right). ( B ) The MCF7 stable cells were infused with Ad-mCherry-GFP-LC3 for 24 h and then treated with 5 μM 4OHT for another 24 h, or treated with 100 nM Baf another 2 h. Then, cells were visualized with a confocal microscope. The autophagosomes (yellow in merge) and autolysosomes (red in merge) in each cell were counted (n = 50 cells/sample). Data obtained from 1 of 3 independent experiments with similar results were presented. Data are presented as the mean±SEM. ***, P< 0.001 (right). ( C ) Transmission electron microscopy images of the MTA1-overexpressing MCF7 cells treated with 5 μM 4OHT for 24 h. Red arrowheads indicate autophagic vacuoles. ( D ) The MTA1-overexpressing MCF7 cells and the control cells were transfected with si ATG7 or treated with 1 μM HCQ. Then cells were treated with 1 μM 4OHT for the indicated time periods and the number of viable cells was counted using a hemocytometer. Cell numbers were presented as the mean±SEM from duplicate plates and data obtained from one of 3 independent experiments with similar results are presented. ***, P< 0.001.

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Microscopy, Transmission Assay, Electron Microscopy, Transfection

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: MTA1 enhances autophagy through activation of the AMPK pathway. ( A ) MCF7 cells transfected with a plasmid encoding FLAG-MTA, the MTA1-overexpressing MCF7 cells, the MCF7/TMAR-1 cells, and their control cells were treated with 5 μM 4OHT for 24 h. Expression levels of protein was analyzed by western blotting. The p-PRKAA level was quantified from band intensity using imageJ and expressed as relative to the level in the parent cells without treatment. The values were normalized by the intensities of the corresponding PRKAA band. Data are presented as the mean±SEM (n = 3). *, P< 0.05, **, P< 0.01, and ***, P< 0.001 (right). ( B ) Quantification of the AMP:ATP ratio by HPLC analysis. The MTA1-overexpressing MCF7 cells, the MCF7/TMAR-1 cells, and their control cells were treated with 5 μM 4OHT for 24 h. Representative HPLC images are shown (top). Data obtained from the HPLC analysis are presented as the mean±SEM (n = 3). *, P< 0.05 and ***, P< 0.001 (bottom). ( C ) Whole cell lysates were obtained from the MTA1-overexpressing MCF7 cells. A mixture of anti-OXPHOS antibodies was used to detect levels of OXPHOS proteins by western blotting. The protein level was quantified from band intensity using imageJ and expressed as relative to the level in the control cells. Values were normalized by the intensities of the corresponding TUBA band. Data are presented as the mean±SEM (n = 3). *, P< 0.05, **, P< 0.01 and ***, P< 0.001 (right). ( D ) The MTA1-overexpressing MCF7 cells or the MCF7/TMAR-1 cells were transfected with si PRKAA for 48 h. At the end of incubation, 20 nM bafilomycin A 1 (Baf) was added at 1 h prior to examination of autophagy flux. The expression of LC3 and NBR1 was determined by western blotting. The LC3-II level was quantified from band intensity using imageJ and expressed as relative to the level in the parent cells without treatment. Values were normalized by the intensities of the corresponding TUBA band. Data are presented as the mean±SEM (n = 3). **, P< 0.01 (bottom). Quantification of NBR1 is shown in Fig. S2B.

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Incubation

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: MTA1 binding peaks are clustered at promoter-transcription start sites with a preference for mitochondrial regulatory genes. ( A ) Genomic location of MTA1-interaction sites. UTR, untranslated region; TSS, transcription start site; TTS, transcription termination site. ( B ) GO term analysis identifies top 15 enriched cellular components derived from 1,533 MTA1 peaks. ( C ) List of 7 genes involved in OXPHOS that are potentially regulated by MTA1. ( D ) Representative ChIP-seq signal of a potential MTA1 target gene, NDUFA10 . ( E ) Total RNA obtained from the MTA1-overexpressing cells and control cells were subjected to qRT-PCR analysis. Expression was normalized using the ACTB gene. Data are presented as the mean fold-change over control±SEM (n = 3). *, P< 0.05 and **, P< 0.01. ( F ) The basal OCR and uncoupled respiration of MCF7 cells that stably expressed MTA1 and control cells. Data presented as mean ± SEM (n = 3). ***P < 0 .001 (left). The basal OCR, and uncoupled respiration were calculated based on data in the left panel. Data presented as mean ± SEM. ***P < 0 .001 (right).

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Control, Quantitative RT-PCR, Expressing, Stable Transfection

Journal: Autophagy

Article Title: MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

doi: 10.1080/15548627.2017.1388476

Figure Lengend Snippet: Level of MTA1 expression correlates with clinical outcome in patients with breast cancer. ( A ) MTA1 expression levels in ESR-positive breast cancer patients who were treated with tamoxifen. The expression levels of MTA1 mRNA in 147 patients with ESR-positive breast cancer treated with tamoxifen for 5 y after surgery (GSE9893) are shown by the log2 expression value (top). The expression levels of MTA1 mRNA in 59 patients with ESR-positive breast cancer treated with standard breast surgery and radiation followed by 5 y of adjuvant tamoxifen (GSE1378) are shown by the log2 expression value (middle). The expression levels of MTA1 mRNA in 136 patients with ESR-positive tumor treated with adjuvant tamoxifen (GSE12093) were shown after normalization and scaled values to a target intensity of 600 (bottom). ( B ) The association of MTA1 expression levels and disease-free survival. The MTA1 low group includes the case with lower MTA1 levels than the median of all patients in the study, and the rest of the patients belonged to the MTA1 -high group.

Article Snippet: The siRNA duplexes targeting MTA1 (5′-AAGACCCUGCUGGCAGAUAAA-3′), ATG7 (5′-GGAGUCACAGCUCUUCCUU-3′), PRKAA (5′-CAAAGUCGACCAAAUGAUA-3′), UQCRC2 (GAGUUUACCAAGUUACCAA) and nonspecific green fluorescent protein (GFP) were synthesized and purified by ST Pharm Co. (Seoul, Korea).

Techniques: Expressing, Adjuvant